THE STABILITY OF ENDOGENOUS TYROSINE-HYDROXYLASE PROTEIN IN PC-12 CELLS DIFFERS FROM THAT EXPRESSED IN MOUSE FIBROBLASTS BY GENE-TRANSFER

Previous studies have used recombinant retroviruses encoding the tyros ine hydroxylase (TH) gene to transduce various cell lines, including f ibroblasts (NIH-3T3), a pituitary tumor cell line (AtT20), and a pancr eatic endocrine line (RIN). These genetically modified cells, synthesi zing either 3,4-dihydroxyphenylalanine, dopamine, or both, are potenti al donors for treatment of Parkinson's disease. However, the levels of TH protein in such transduced cells have been low and heterogeneous. Using several modified versions of retrovirus vectors encoding TH, we demonstrate that protein stability is an important factor governing le vels of TH in NIH-3T3 fibroblasts. Whereas low levels of TH protein we re observed in infected NIH-3T3 cells, high levels of a TH-beta gal fu sion protein were found. This difference was due to a significantly lo nger half-life of the TH-beta gal fusion protein relative to TH alone. However, the TH-beta gal fusion protein was found to be enzymatically inactive. We also found that the half-life of the endogenous TH prote in in PC-12 cells is sevenfold longer than the TH protein in transduce d fibroblasts, implying that a cell-type specific regulator or mechani sm may stabilize TH in catecholaminergic cells.